A SNaPshot assay for the rapid and simple detection of known point mutations conferring resistance to ACCase‐inhibiting herbicides in Lolium spp.

Abstract

Summary Evolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non‐target‐site‐based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high‐throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance‐associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase‐inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96‐ or 384‐well plate formats, thus reducing work time and cost.

Resistance to acetyl‐CoA carboxylase‐inhibiting herbicides

“…Therefore, the availability of rapid early diagnostics tools for confirming product failures is imperative for slowing down the evolution of resistance to ACCase herbicides. The discovery of precise nucleotide changes involved in target‐site resistance and the growing accessibility of molecular biology techniques have allowed development of a wide range of methodologies for detecting the known resistance mutations . However, these methods have rarely been used outside research owing to the high costs involved, restrictions to target‐site resistance only and ambiguity arising from multiple amino acid changes at specific resistance codon positions .…”

“…Analysis of a large number of Lolium spp. populations from the United Kingdom and Australia showed a predominance of the D2078G and I2041N mutations respectively, potentially reflecting the alternating use of FOPs and DIMs in small‐grain cereal and dicotyledonous break crops in the United Kingdom and a more prolonged FOP use in continuous wheat cropping systems in Australia. Importantly, DNA analysis from a few hundred herbarium blackgrass plants predating any synthetic herbicide use revealed the presence of an I1781L mutation in one of the individuals tested, thus indicating that some ACCase mutations are intrinsically more prevalent than initially assumed …”

Assessing Genetic Variation and Spread of Phalaris minor Resistant to ACCase Inhibiting Herbicides in Iran

“…Lolium spp. populations from the United Kingdom and Australia showed a predominance of the D2078G and I2041N mutations, respectively (Alarcón-Reverte et al, 2013;Malone et al, 2014) The remaining resistant populations from the Fars region clustered together. In these populations, resistance to ACCase inhibiting herbicides is not target site related (data not shown).…”

Investigating target‐site resistance mechanism to the PPO‐inhibiting herbicide fomesafen in waterhemp and interspecific hybridization of Amaranthus species using next generation sequencing

“…22 To investigate herbicide resistance mechanisms and screen known resistance-endowing mutations, several molecular techniques have been used for many years including: Sanger sequencing, CAPs, dCAPs, allele-specific polymerase chain reaction (PCR), Taqman, Multiplex SNaPshot, and Scorpions. [28][29][30][31] These methods are limited in that screening is performed for a specific mutation, and is costly and time-consuming given the large number of samples often involved in herbicide-resistant weed surveys. By comparison, next-generation sequencing (NGS) methods due to the depth of reads, can be used more effectively on large pooled samples compared with other techniques.…”